Toll-Like Receptor 4 activation and function in diseases: an integrated chemical-biology approach.

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 642157

“Kick-off meeting” February 11-12, 2015, Milan


Based on an international team derived from the COST action BM1003 (, 2011-2014) and thus relying on consolidated group interactions and synergies and on a unique combination of chemistry, biology, biophysics, biochemistry and pharmacology expertise, the TOLLerant project aims to gain information on molecular aspects of TLR4 activation and signalling by using synthetic and natural compounds and nanoparticles that interact selectively with some components (mainly MD-2 and CD14) of the TRL4 recognition system.
TLR4 is an emerging molecular target related to an impressively broad spectrum of modern day disorders still lacking specific pharmacological treatment. These include autoimmune disorders, chronic inflammations, allergies, asthma, infectious and CNS diseases, and cancer.
The short-term scientific objective is to develop novel, non-toxic, synthetic and natural TLR4 modulators (agonists or antagonists) and to assess their therapeutic potential on animal models of TLR4-related acute and chronic pathologies. The long-term scientific objective is to develop a new generation of innovative, TLR4-based therapeutics, to be used as vaccine adjuvants, anti-sepsis agents, and anti-inflammatory agents to treat chronic inflammations (allergy, asthma). An obstacle in achieving these high impact goals is the lack of suitably trained scientists with broad skills and understanding of the underpinning basic life science and cutting-edge chemistry. The training programme will fill this gap and provide Early Stage Researchers (ESR) with broad competences, experience and skills in the cutting-edge, inter-disciplinary research in the field of chemical biology related to the molecular mechanisms of innate immunity and inflammation. During the training, the young researchers will be supported by senior scientists to cultivate their scientific, entrepreneurial and inter-cultural mindset. The non-academic sector will be committed to provide ESRs with entrepreneurship and company management skills, in order to enhance their employability by the private sector or even to motivate them to create own start-up companies.

Research methodology and approach

The research programme will be developed according to the following work packages (WPs):

WP1. DESIGN, SYNTHESIS OF NEW TLR4-ACTIVE COMPOUNDS, STRUCTURAL STUDIES ON POTENTIAL TLR4 MODULATORS FROM BACTERIA AND PLANTS: Starting from structure-activity data from previous generations of TLR4-active cationic and anionic amphiphiles developed by UNIMIB partner, new synthetic compounds will be rationally designed (CSIC) and synthesized (UNIMIB).
The rational design will rely on chemical structures of existing ligands. Ligand-based design will be integrated with structure-based design and virtual screening approaches to create novel, high affinity ligands for MD-2 and CD14 co-receptors (CSIC). These compounds will act as TLR4 blockers (antagonists) or activators (agonists), and they could also be considered for the exploitation as TLR4 probes with the appropriate chemical modifications (insertion of fluorescent moieties and other chemical labels, such as triple bonds for Raman scattering, see WP2).
These ligands cannot be tracked in vivo and therefore it is not possible to apply molecular imaging techniques in the drug development process. The possibility of combining precise in vitro and/or in vivo delivery with accurate imaging is considered important for clinical development, as it can maximize the effectiveness of drugs, minimise the invasiveness and toxic side effects and speed up the clinical development program. To address these problems, TLR4 ligands developed in WP1 will be assembled on nanoparticles (NP), taking advantage of the unique physicochemical properties of the NP and the protocols developed at BIOMAGUNE specifically for NP radiolabelling to monitor in vivo trafficking patterns and activity in real time using complementary, non-invasive molecular imaging techniques such as high field magnetic resonance imaging (MRI), positron emission tomography (PET) and single-photon emission computed tomography (SPECT). TLR4 agonists will be also coupled to polypeptide-based NP (KI), and to Polyglutamic acid (PLE) and hyaluronic acid (HA) polymers and combined with protein antigens/allergens (Lofarma), including hypoallergenic recombinant mutant allergens in order to prepare efficient vaccines and immunotherapeutics.
In parallel, the chemical structure of lipid As from Gram- bacteria and of TLR4-active natural compounds from various sources will be investigated by a combination of chemical and spectroscopy approaches (UNINA, DIOMUNE). This important study will parallel the rational design/synthesis of TLR4-active compounds with the scope to enlarge the repertoire of small molecule chemical structures (scaffolds) available to TLR4 modulation. The lipid A variants and natural compounds will be: 1) scaffolds for the synthesis of new compounds, 2) hit compounds for TLR4 modulation to be studied in WP 2. Results from WP2 will be integrated in an iterative process to undertake the elucidation of the mechanistic aspects of the TLR4 activation/inactivation by means of computational techniques (CSIC, MIT).

WP2. IN VITRO LIGAND/RECEPTORS INTERACTIONS STUDIES; EXPERIMENTS ON CELLS. The interaction between synthetic and natural molecules and the different components of the TLR4 recognition system (MD-2, MD-2.TLR4 complex and CD14) will be studied in vitro by an array of complementary techniques: NMR binding studies on purified proteins (CSIC), binding experiments on cells with opportunely labelled ligands (UNIBI).
Pure, functional proteins CD14 and MD-2 for NMR studies will be produced by the KI partner.
The co-localization of TLR4 agonists and antagonists with the receptor CD14 and with the activated complex TLR4.MD-2, will be studied by imaging techniques in excised tissue sections either fixed or during perfusion experiments by two-photon fluorescence microscopy and/or coherent anti-Stokes Raman scattering (CARS) microscopy (UNIBI).
The agonist/antagonist activity of all molecules will be assessed on cellular models endogenously expressing TLR4, CD14 and MD-2 (e.g. macrophages and epithelial cells, KI and VIB) or cells engineered with a gene reporter system that reveals the activation of NF-kB and IRF-3 by active TLR4 signalling (HEK-blue cells-TLR4 or cells transiently transfected with NF-κB and IRF-3 dependent luciferase reporter genes; UNIMIB and VIB). The influence of TLR4 modulation on downstream NF-κB and IRF3-dependent gene expression will also be investigated by endogenous gene expression profiling via Q-PCR, ELISA, Bio-Plex assays, or cytokine bioassays (VIB).
The TLR4 agonists conjugated to NPs, proteins, and PLE and HA polymers, will also be checked by in vitro human IgE-system to evaluate their immunogenic potential (Lofarma).
All the results will be rationalized with help of molecular modelling to re-direct the design and optimization of future ligands (FUSP-CEU, UNIMIB). Mutants of MD-2, MD-2.TLR4 and CD14 will be proposed and considered in order to validate hypothesis related to the involved mechanisms underlying the molecular recognition events.

The biological and pharmacological effects of TLR4 modulation of molecules and NP will be studied in three animal models, in the perspective to realize a complete pre-clinical phase. The molecules with best activity as TLR4 agonists (WP2), conjugated to NP or PLE and HA polymers, will be tested in vivo as anti-allergy vaccine adjuvants (Lofarma). The more potent antagonists will be tested on animal models of sepsis (DIOMUNE, VIB) and asthma (VIB).
The results obtained in the in vitro and in vivo phases will be used in an iterative way to improve, respectively, target affinity and pharmacokinetic properties (adsorption, distribution, bioavailability, toxicity) of the new molecules. This multi- and interdisciplinary work requires a tight connection among different sectors and expertise in the network and a continuous and bidirectional information flow from the design/synthesis to the in vitro and finally the in vivo experiments. This reduces the cycle time of each phase of drug discovery and development and minimizes hazards and risks early in the design through advanced molecular imaging studies that will ensure rapid development and identification of the best drug candidates.
The individual research programs of recruited Early Stage Researchers (ESR) will be focused on one or more of the above-mentioned WPs and integrated into the overall research program. Different and complementary experimental approaches and methods ranging from computational chemistry to synthetic and medicinal chemistry to biochemistry, immunology and biophysics will be used in the project and transferred to ESRs.